Association between Long Noncoding RNA ANRIL Expression Variants and Susceptibility to Coronary Artery Disease

Animal cells possess thousands of long non-coding (lnc) RNAs, such as antisense noncoding RNA in the INK4 locus (ANRIL), which have regulatory roles in the cells’ molecular mechanisms, including X-chromosome inactivation, and developmental processes. These lnc RNAs are known to influence the extensive spectrum of age-related disorders. Accordingly, there is evidence for the role of these lnc RNAs in cardiovascular diseases, particularly coronary artery diseases (CAD). The aim of this study was to assess whether the expression of the lnc RNA ANRIL was associated with a susceptibility to CAD by evaluating the expression level of the two transcripts of ANRIL. Peripheral blood was taken from fifty patients affected by CAD and relative expression of ANRIL was determined by Real-Time PCR assay. The obtained data indicated that the EU741058 transcript expression level significantly decreased in CAD patients in comparison with the healthy individuals (P= 0.001). Furthermore, there was no significant association between the NR_003529 transcript expression, and CAD risk in Iranian patients (P=0.751). Our results suggest that the expression level of the EU741058 transcript of ANRIL may be implicated in CAD development, creating a predictive biomarker for CAD patients in future.

ardiovascular disease (CVD) is known to be one of the main causes of human death worldwide (1). In 2012, the World Health Organization (WHO) has reported that ischemic heart disease has been mentioned as the first leading cause of mortality in the world (2). A cohort study on the Iranian population indicated that there was a high incidence of cardiovascular disease (CVD), and mortality in both sexes in Iran other populations (4)(5)(6)(7). The chromosome region 9p21 has been reported to be an important susceptibility locus for several multifactorial diseases including CAD, ischemic stroke, aortic aneurysm and type 2 diabetes mellitus (8).
Long non-coding RNAs (lncRNAs) have been implicated in many important biological mechanisms, in particular imprinting, histone-code regulation, gene regulation, and cell proliferation. Several studies have indicated that the lncRNA expression level is associated with manifesting some disorders including atherosclerosis (9)(10)(11) (13,16,17).
Functional analysis of the 9p21.3 region has revealed that there might be a functional enhancer that influences the expression of ANRIL variants, NR_003529 and EU741058 (15). The previous studies showed that there was a significant association between risk alleles of ANRIL gene and risk of atherosclerosis (12,18). Furthermore, the expression of ANRIL variants have been shown to be different between patients affected by atherosclerosis and healthy individuals (17).
The methylation of the ANRIL target, p15 INK4b , has been found to be associated with the expression of the EU741058 variant of the ANRIL gene in CAD patients (19). In the present study, we aimed to assess whether ANRIL expression variants were associated with the susceptibility to CAD in Iranian patients. The results obtained from this study could improve our understanding of the molecular mechanisms involved in the manifestion of CAD.

RNA isolation and cDNA synthesis
The peripheral venous blood was taken in the morning from patients fasting after midnight, and the total RNA was extracted by QIAamp RNA Blood Mini kit (Qiagen, Germany). After determining the concentration of RNA, the cDNA synthesis was carried out using a QuantiTect Reverse Transcription kit (Qiagen, Germany).

Quantitative reverse-transcript polymerase chain reaction (qRT-PCR)
The quantification of the relative expression was performed in triplicate for each sample. The beta actin was used as the reference gene to normalize the expression level of the ANRIL gene.
The sequence of primers and probe were presented in Table 1. The expression level was determined using Premix Ex TaqTM (Takara Biotechnology, Tokyo, Japan) according to the manufacture's guidelines. An initial denaturation was performed at 95 °C for 30 s followed by 45 cycles of 95 C for 10 s, and 60 C for 30 s. The PCR products were confirmed using a 2% agarose gel.

Statistical analysis
All statistical analyses were performed using The area under the ROC curve was used to evaluate its overall diagnostic accuracy.

Results
The demographic and biochemical characteristics of patients did not show a significant difference compared with the studied healthy individuals (Tables 2 and 3). However, most of the individuals of the present study were urban residents ( Table 3).
The study population comprised 68 males and 32 females with the mean age of 53 years ( Table 2).